A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca2+-Triggered Millisecond Exocytosis

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• 作者：Paul Heo ; Yoosoo Yang ; Kyu Young Han ; Byoungjae Kong ; Jong-Hyeok Shin ; Younghoon Jung ; Cherlhyun Jeong ; Jaeil Shin ; Yeon-Kyun Shin ; Taekjip Ha ; Dae-Hyuk Kweon
• 刊名：Journal of the American Chemical Society
• 出版年：2016
• 出版时间：April 6, 2016
• 年：2016
• 卷：138
• 期：13
• 页码：4512-4521
• 全文大小：695K
• ISSN：1520-5126

文摘

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca²⁺ stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca²⁺. When 10 μM Ca²⁺ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.